Decellularized silk fibroin scaffold primed with adipose mesenchymal stromal cells improves wound healing in diabetic mice

Introduction:
Silk fibroin (SF) scaffolds have been shown to be a suitable substrate for tissue engineering
and to improve tissue regeneration when cellularized with mesenchymal stromal cells
(MSCs). We here demonstrate, for the first time, that electrospun nanofibrous SF patches,
cellularized with human adipose-derived MSCs (Ad-MSCs-SF) or decellularized (D-Ad-
MSCs-SF) are effective in the treatment of skin wounds, improving skin regeneration in
db/db diabetic mice.
Methods:
The conformational and structural analyses of SF and D-Ad-MSCs-SF patches were
performed by scanning electron microscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. Wounds were performed by a 5mm
punch biopsy tool on the mouse’s back. Ad-MSCs-SF and D-Ad-MSCs-SF patches were
transplanted and the efficacy of treatments was assessed by measuring the wound closure
area, by histological examination and by gene expression profile. We further investigated the
in vitro angiogenic properties of Ad-MSCs-SF and D-Ad-MSCs-SF patches by affecting
migration of human umbilical vein endothelial cells (HUVECs), keratinocytes (KCs) and
dermal fibroblasts (DFs), through the aortic ring assay and, finally, by evaluating the release
of angiogenic factors.
Results:
We found that Ad-MSCs adhere and grow on SF, maintaining their phenotypic mesenchymal
profile and differentiation capacity. Conformational and structural analyses on SF and D-Ad-
MSCs-SF samples, showed that sterilization, decellularization, freezing and storing did not
affect the SF structure. When grafted in wounds of diabetic mice, both Ad-MSCs-SF and DAd-
MSCs-SF significantly improved tissue regeneration, reducing the wound area
respectively by 40% and 35%, within three days, completing the process in around 10 days
compared to 15-17 days of controls. RT2 gene profile analysis of the wounds treated with Ad-
MSCs-SF and D-Ad-MSCs-SF showed an increment of genes involved in angiogenesis and
matrix remodelling. Finally, Ad-MSCs-SF and D-Ad-MSCs-SF co-cultured with HUVECs,
DFs and KCs, preferentially enhanced the HUVECs’ migration and the release of angiogenic
factors stimulating microvessel outgrowth in the aortic ring assay.
Conclusions:
Our results highlight for the first time that D-Ad-MSCs-SF patches are almost as effective as
Ad-MSCs-SF patches in the treatment of diabetic wounds, acting through a complex
mechanism that involves stimulation of angiogenesis. Our data suggest a potential use of DAd-
MSCs-SF patches in chronic diabetic ulcers in humans.